JPH02309241A - Adrenalin detection sensor - Google Patents

Abstract

PURPOSE:To make the title sensor inexpensive and to enhance the capacity thereof by using a measuring pH-ISFET covered with an adrenalinmonoamine oxidase imiobilized film containing flavin. CONSTITUTION:A drain 2 and a source 2' are formed on a P<->-type silicon substrate and drain and source electrodes 5, 6 are formed thereon by vapor deposition. An SiO2 film 7 is formed to the surface of an element by thermal oxidation and an Si3N4 film 8 is formed thereto by a CVD method. An adrenalinmonoamine oxidase immobilized film 10 containing flavin reacting only with adrenalin is provided and the part other than a gate part is molded by a resin mixture 9. This electrode is used as a detection electrode and an electrode having the same structure as said electrode but no adrenalinmonoamine oxidaze immobilized film is used as a comparing electrode.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is applicable to acetylcholine, dopamine, serotonin,
Even if other substances such as r-aminobutyric acid and glutamic acid are mixed, only adrenaline is felt, and it is attracting attention as one of the substances related to senile dementia, and the concentration of adrenaline present in adrenal medulla cells, brain and peripheral nerve cells. This invention relates to an adrenaline detection sensor that detects adrenaline.

Conventional technology The detection of adrenaline, which has been attracting attention as one of the substances associated with senile dementia, is currently being conducted by extracting human brains from the spinal fluid and finding that this substance is reduced in Parkinson's disease patients. Conventionally, liquid chromatography has been used as a separation method, and amberometry, which is an electrochemical analysis method, has been used as a quantitative method, and these have been used in combination. Liquid chromatography performs separation by utilizing the time difference between the components flowing out due to the difference in adsorption power by the adsorbent in the column, and the separated components are introduced into an electrochemical detector. When applying a DC T2C pressure of about OV with the working pole as the anode, the current flowing is the fifth
As shown in the figure, this current is due to the oxidation of catecholamines and their metabolites, including adrenaline, and is quantified by utilizing the fact that their peaks are proportional to their respective concentrations.

Problems to be Solved by the Invention As mentioned above, conventional methods involve extracting, separating, and quantifying cerebrospinal fluid from a living body, and do not directly measure local information within the body. If we were to obtain internal information using this method, the device would have to be made smaller.
It's virtually impossible. Furthermore, there is no guarantee that passing an electric current directly through a specimen is safe for the human body.

In addition, the detection equipment using liquid chromatography/electrochemical analysis is large, and in order to prevent external temperature changes, the anherometry detector is placed in a constant temperature room, and the pre-heater for the detector is kept strictly at 10.04℃ or less. Since temperature stability must be maintained, temperature control is complicated and, therefore, the cost is high.

In order to eliminate the drawbacks mentioned in the conventional method, the present invention reacts only with adrenaline to form 3-4-dihydroxymandelic acid in proportion to the concentration of adrenaline by the action of flavin-containing adrenaline monoamine oxidase. The object of the present invention is to provide an adrenaline detection sensor that measures pH changes using a potentiometry method, is small, easy to measure, and inexpensive. Note that this detection sensor can also perform measurements in vivo.

Means for Solving the Problems In order to achieve the above object, the technical solution of the present invention is as follows:
Measurement pH-15F covered with a membrane containing flavin-containing adrenaline monoamine oxidase immobilized on one substrate
Prepare two types: an ET (ion-sensitive field effect transistor) and a reference pH-I S FET without an immobilization film.
By compensating for environmental temperature and pH, it senses pure adrenaline and indicates its concentration.

Effects According to the present invention, even if various substances are present, only adrenaline is decomposed by flavin-containing adrenaline monoamine oxidase to form 3-4-dihydroxymandelic acid. As a result, the pH inside the membrane changes depending on the concentration of adrenaline present outside. Then, the pressure changes as E = (RT/F)・1ogpH according to the Nernst equation, where R is the gas constant, T is the feed temperature, F is the Faraday constant, and the channel of F]liT electrons are induced, and a current flows between the source and drain, and this current changes even if the external solution pH changes or the temperature changes, so a pH-FBT that is sensitive only to the electrolyte pH and temperature is installed in parallel. This is compensated by a differential circuit to detect only the concentration of adrenaline in the solution.

Example Embodiments of the present invention will be described in detail below with reference to the drawings.

Figure 1 <a> is a top view of the measured pH-15FET (4-on sensitive field effect transistor), and Figure 1 (b)
is (a)! It is a sectional view at -1'. 1 is a P-type silicon substrate. 2.2' is a drain and source n+ region formed by doping phosphorus thereon by diffusion.

3 is a P-type channel portion. 4 is a P channel stopper in which boron is diffused and formed on the surface other than the gate portion at the tip. 5 is the drain electrode made of Au-
Cr is vapor-deposited. 6 is the source electrode made of Au-C
It has been vapor-deposited with r. 5.6 is the drain, respectively.
This is the source electrode Aj terminal. 7 is an 8i02 film formed on the element surface by thermal oxidation. This is a Si3N4 film formed by the 8Fi, C■ method. 9 is a mixed resin of epoxy and silicon which is molded except for the gate portion. 10 is a flavin-containing adrenaline monoamine oxidase-immobilized membrane that reacts only with dopamine.

Figure 2 shows the configuration of the reference pH-l5FE'I' (ion-sensitive field effect transistor), where Figure 2 (a) is a plan view and Figure 2 (b) is 1-1 in Figure 2 (a). FIG. The reference pH-15F'B'r has almost the same configuration as the measurement pH-18FET shown in FIG. 1, and differs in that no immobilization film is provided at the gate portion.

Next, we will describe a method for immobilizing flavin-containing adrenaline monoamine oxidase onto a membrane.
The pH was adjusted to 7 by adding HcJ, and the gate electrode was immersed in a water bath heated to 50°C for 2 hours to react.
Chemically modify 3N4. The chemically modified gate electrode was immersed in a solution containing 250 mg of cellulose triacetate dissolved in 10 rM dichloromethane, 100 μl of 50% glutaraldehyde and 500 μl of 4-aminomethyl-1,8-octanediamine, and then placed in a desiccator. Store it for one day to allow the crosslinking reaction to proceed. Furthermore, aldehyde groups were introduced onto the surface of the organic film by reacting with 100% glutaraldehyde solution at room temperature for 1 hour, and after thorough washing with a pH 7 phosphate buffer, the sapphire substrate pH-F
Flavi the ET gate at 4°C.

The flavin-containing adrenaline monoamine oxidase is immobilized with gK by substituting the amino group and the aldehyde group by immersing it in a saturated solution in which the adrenaline monoamine oxidase contained therein is dispersed.

FIG. 3 is a measurement circuit diagram of the pH-15FET for detecting adrenaline.
A constant drain current Id always flows through II, and a constant voltage Vd=RI1 is applied between the source and drain of the pH-15FBT 11 for measurement by another current source 18. The interfacial potential changes due to pH changes in the solution.
Constant pH-15F'BT 11 (7) Id, Vd7
) Since f cannot change, the potentials between the source and drain both change by the amount of the interface potential change l), and this change is output to Voutz. In order to improve the response characteristics to changes in the concentration of the solution and uniform pH changes, the reference pH-l
5FET K outputs Voutz cell.

24Fi is a differential amplifier for taking the difference.

The measurement method is to first set a small micro ring,
A small amount of body fluid (amount Fi, 0.5 μl) was taken out into this, and the measurement p, H-ISFET11 with flavin-containing adrenaline monoamine oxidase immobilized and the bare pH-15FET without immobilization (reference 15FET)
The surfaces of Ag and Ag are chlorinated and the three electrodes of Ag/AgC1 reference electrode 13 are brought into liquid junction. Operational amplifier 14.15.
16.17 Always 1 to painter 5FET11.12 by
A drain current of 0 μA is caused to flow, and a constant DC voltage (1 to 2 V) is applied between the source and drain through the current source 18, 19, 20, 21 and the resistor 22, 23.

Then, the gate insulating part of each pH-15FET detection sensor is psio2 according to the principle of I5FET.
At the interface between the insulating layer and the electrolyte, an interfacial potential is generated due to the ion concentration, and this is applied to the silicon surface, changing the drain electrode.

Then, the differential circuit changes the temperature change and pH of the measurement solution.
The changes are compensated for and an output dependent only on the concentration of adrenaline is obtained by the differential amplifier 24, resulting in an output result as shown in FIG.

Effects of the Invention As described above, the effects of the present invention are as follows: 1) Measurement using H-ISFET and reference pH- without the immobilized membrane on one substrate. Prepare two l5FETs,
This enzyme is compact, easy to measure, inexpensive, unaffected by temperature and pH changes of the solution, does not require temperature compensation, and can be used even in the coexistence of various types of substances. The selectivity makes it possible to provide a detection sensor that only acts on adrenaline. Note that this detection sensor is also capable of measuring in vivo (local information within a living body can be directly measured using a current between a source and a drain).

[Brief explanation of drawings]

FIG. 1(a) is a plan view of adrenaline measurement pH-15F'ET in one embodiment of the present invention, FIG. 1(b) is a cross-sectional view on the I-1' plane of the same figure <a>, and FIG. (a,)
is a plan view of the reference pH-18FET in this example, and FIG. 2(b) is a cross-sectional view taken along the plane 1-1' of FIG.
Figure 3 shows pH-15FE for adrenaline detection in this example.
Figure 4 is a diagram showing the relationship between the concentration of adrenaline and current between source and drain. Figure 5 is a diagram showing the relationship between current value and concentration due to oxidation of catecholamines and metabolites including adrenaline in the conventional method. . 1...P-type silicon, 21 Drai znM region, 2...
・:/-sn lJ region, 3... P- type channel, 4... Channel stopper (P), 5... Source electrode, 5... Source electrode AJ terminal, 6...・Drain electrode, 6,...Drain electrode AJ terminal, 7...5i
02.8...Si3N4. 9... Mixed resin of epoxy and silicone, 10... Adrenaline monoamine oxidase immobilized membrane containing flapine, 11... 2 measurement pH-FET. 12...Reference pH-FET, 13...Ag/Ag
C1 reference pole, 24...differential amplifier. Name of agent: Patent attorney Shigetaka Awano and one other person Figure 1 (α) 6, t-x*heel tOTa': i! , it=S Fig. 2 (Q) (b) Fig. 3 fδ

Claims (1)

[Claims] pH - measurement p coated on the gate of a field effect transistor with a membrane immobilized with flavin-containing adrenaline monoamine oxidase, which reacts with adrenaline and changes the pH.
An adrenaline detection sensor comprising an H-field effect transistor and an uncovered reference pH-field effect transistor, and differential means for both pH-field effect transistors.